Abstract

5666

Polycyclic aromatic hydrocarbons (PAH) are likely to play a major role in cancer induction in smokers and certain occupationally exposed groups. Humans vary widely in their abilities to metabolically activate or detoxify PAH, and these variations may be related to cancer risk. Many studies have examined the possible relationship between variants in genotypes of carcinogen metabolizing enzymes such as glutathione-S-transferases and cancer risk. Phenotyping may be a better way to determine inter-individual differences in PAH metabolism. Our laboratory has developed methods for analysis of phenanthrene metabolites in urine, as an approach to PAH metabolite phenotyping. Methods to measure metabolic activation by the diol epoxide pathway and detoxification by phenol formation have already been published. In this study, we developed a method to quantify Phe-epoxide-NAC, the urinary meracapturic acid end-product of glutathione conjugation of phenanthrene-9,10-epoxide, and compared levels of this metabolite to variants in glutathione-S-transferase genotypes. [D10]Phe-epoxide-NAC was used as internal standard. Urine samples were purified by passage through cation exchange, anion exchange, and reverse phase solid phase extraction cartridges. Analysis and quantitation was achieved using liquid chromatography-electrospray ionization-tandem mass spectrometry with selected reaction monitoring, in the negative ion chemical ionization mode. The transition m/z 356 (M − 1)− → m/z 209 [M − (H2O+ CH2CH(COOH)NHCOCH3)] was monitored. Clean peaks corresponding to two diastereomers of Phe-epoxide-NAC were observed in 15 urine samples while none were detected in another 17. Levels of this metabolite varied from not detected (< 20 fmol/ml) to 877 fmol/ml. Mean levels of Phe-epoxide-NAC in smokers (243 fmol/ml urine, N = 38) were not different from those in non-smokers (296 fmol/ml, N = 6). Levels of Phe-epoxide-NAC (457 ± 280 fmol/ml, N = 8)) were substantially higher in GSTM1 present than in GSTM1 null individuals (15.8 ± 30.1, N = 22). Levels of this metabolite were also higher in individuals with both GSTM1 and GSTT1 present than in those with both GSTM1 and GSTT1 null. These results suggest that GSTM1 and GSTT1 presence or absence do affect levels of PAH detoxification through the mercapturic acid pathway. This is the first report of quantitation of a PAH mercapturic acid in human urine thus allowing a phenotype-genotype comparison for this important carcinogen detoxification pathway.